Method for decreasing cartilage damage in dogs

ABSTRACT

A method for modulating enzymatic degradation of articular cartilage in a dog comprises administering to the dog an enzymatic degradation modulating effective amount of eicosapentaenoic acid (EPA), for example as a component of a food composition. By practice of the method in a dog having arthritis, mobility of the dog can be increased, weight bearing in an arthritic limb can be increased, and/or pain associated with arthritis can be reduced.

This application is a continuation in part of U.S. patent applicationSer. No. 10/912,864, filed Aug. 6, 2004, which claims priority of U.S.patent application Ser. No. 10/638,832, filed Aug. 11, 2003 andconverted to a provisional application, Ser. No. 60/608,926, on Aug. 5,2004. The above-cited applications are incorporated herein by referencein their entirety.

FIELD OF THE INVENTION

The present invention relates to methods of managing arthritis andarthritis-related conditions in companion animals, more particularlydogs.

BACKGROUND OF THE INVENTION

Arthritis, more particularly osteoarthritis, is a degenerative jointdisease commonly occurring in humans and in companion animals. See forexample Richardson et al. (1997), Vet. Clin. North Amer. Small AnimalPractice 27, 883-911.

Osteoarthritis involves progressive deterioration of articularcartilage, with loss of proteoglycan and collagen and proliferation ofnew bone, accompanied by a variable inflammatory response within thesynovial membrane. It is the most common form of joint andmusculoskeletal disease affecting dogs but is relatively uncommon incats. See for example Schoenherr et al. (2000) in Hand et al., eds.:Small Animal Clinical Nutrition, 4th ed., 907-921, Walsworth PublishingCo., Marceline, Mo.; Hedborn et al. (2002) Cell Mol. Life Sci. 59,45-53; Pool (1999) Front. Biosci. 4, D662-D670.

Management of osteoarthritis can include pharmacological treatments,surgery, nutraceutical administration and diet management. Such currentmanagement approaches have, however, focused on symptomatic relief andas such they have not been entirely successful in disease management orin treating the underlying pathologies. Hence there remains a continuingneed for new approaches in managing osteoarthritis in companion animals,more particularly dogs.

Omega-3 (also known as n-3) fatty acids are needed in diets of mammals.They are naturally occurring materials in foods and have been used indietary supplements. Schoenherr et al. (2000), supra, reviewed use offatty acids including n-3 fatty acids in inflammatory disease includingarthritis, and referenced a compilation by Miller et al. (1992) CaninePractice 17(6), 6-8, of observations of dog owners who perceivedimprovement in clinical signs of arthritis in their dogs when treatedwith fatty acids for dermatological problems.

Three omega-3 fatty acids are currently of most interest as dietarycomponents: eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) andalpha-linolenic acid (ALA). Hitherto no great distinction has been drawnamong these three.

SUMMARY OF THE INVENTION

It has now been found that, in the canine, omega-3 fatty acids are notequal in their activity in arthritic conditions where cartilage isinvolved. In studies reported herein, only EPA, but not DHA or ALA, wasabsorbed appreciably by canine cartilage, and cartilage damage asmeasured by glycosaminoglycan (GAG) release was significantly loweredafter exposure to EPA, but not DHA or ALA. Benefits of administering thespecific omega-3 fatty acid EPA to a dog having arthritis can includeincreased mobility of the dog, increased weight bearing in a limb of thedog, and reduced pain associated with the arthritis.

Accordingly there is now provided a method for increasing mobility of adog having arthritis, the method comprising administering to the dog EPAin an amount effective to decrease cartilage damage in the dog.

There is further provided a method for increasing weight bearing in alimb of a dog having arthritis, the method comprising administering tothe dog EPA in an amount effective to decrease cartilage damage in thedog.

There is still further provided a method for reducing pain associatedwith arthritis in a dog, the method comprising administering to the dogEPA in an amount effective to decrease cartilage damage in the dog.

In a further embodiment of the invention, there is provided a method formodulating enzymatic degradation of articular cartilage in a dog, themethod comprising administering to the dog an enzymatic degradationmodulating effective amount of EPA.

According to the above methods, the EPA can be administered by variousroutes, including orally as a component of a food composition.

Further advantages and benefits of the present invention will beapparent to one skilled in the art from reading this specification.

DETAILED DESCRIPTION

This invention involves administration of EPA as a method of managingosteoarthritic diseases and conditions, and symptoms thereof, in dogs.

Omega-3 fatty acids are a recognized group of polyunsaturated long-chain(generally 12-26 carbon atoms) carboxylic acids. The physiologicallymore important omega-3 fatty acids have unbranched chains that are 18-22carbon atoms in length. All have a double bond between the 3rd and 4thcarbon atoms as counted from the methyl (omega) end of the molecule.Eicosapentaenoic acid (EPA) has a chain length of 20 carbon atoms andhas a total of five double bonds, including one at the omega-3 position.

When an omega-3 fatty acid, in particular EPA, is mentioned herein, itwill be understood that derivatives thereof, known to those of skill inthe art, can be substituted if desired. Examples of suitable derivativesinclude esters, such as branched or unbranched and/or saturated orunsaturated C₁-C₃₀ alkyl or cycloalkyl esters, in particular C₁-C₆ alkylesters, of omega-3 fatty acids, particularly EPA.

EPA can be administered to a dog by one or more of many routes ofadministration, such as, for example, oral, intranasal, parenteral(e.g., intravenous or subcutaneous) routes and the like. The oral routeis particularly suitable and EPA can be administered orally in anutraceutical or pharmaceutical dosage form or as a component of a foodcomposition.

When present in a food composition, which can be wet or dry, EPA can beincorporated therein, for example by any suitable mixing procedure,and/or distributed on the surface of food pieces, for example byspraying, agglomerating, dusting or precipitating on the surface. Inparticular embodiments EPA is present in a food composition providingthe nutritional diet per se, in a snack, supplement or treat, or in aliquid portion of the diet such as water or another fluid.

EPA can alternatively be administered in solid form such as a powder, orin liquid or gel form, or in a nutraceutical or pharmaceutical dosageform such as a capsule, tablet, caplet, syringe or the like. Within sucha dosage form the EPA can be present in solid, liquid or gel form. Anyof the usual nutraceutical or pharmaceutical carriers can be employedtogether with the EPA, including water, glucose, sucrose and the like.

In certain embodiments, EPA-containing food compositions areadministered that are essentially free of DHA and/or ALA. “Essentiallyfree of DHA and/or ALA” is intended to mean that either or both of DHAand ALA are substantially absent or that there are only small andinsignificant amounts of either or both of DHA or ALA present, forexample, less than about 0.1%, less than about 0.03%, less than about0.01%, less than about 0.003% or less than about 0.001%, by weight ofthe composition. In embodiments that are “essentially free of DHA and/orALA” herein, any amount of DHA and/or ALA present is at a concentrationsufficiently low so that no substantial incremental effect is producedin an osteoarthritic dog on osteoarthritis or the progression thereof orsymptoms produced thereby.

In other embodiments, present with the EPA can be other omega-3 fattyacids such as DHA and ALA in significant quantities. In someembodiments, omega-6 fatty acids such as linoleic acid, gamma-linolenicacid (GLA) and/or especially arachidonic acid (AA), can also be present.Omega-3 and omega-6 fatty acids can be found in sources such as fishoils and fish meals in relatively large quantities. According to thepresent invention, the benefits in decreasing cartilage damage byadministration of a mixture of omega-3 fatty acids, or a mixture ofomega-3 and omega-6 fatty acids, are attributable largely or essentiallywholly to EPA. In any such mixture, therefore, it is important that EPAbe present in an amount effective to decrease cartilage damage in a dog.

EPA administered according to the present method is effective againstvarious forms of osteoarthritis as well as other forms of arthritisincluding rheumatoid arthritis.

EPA acts to inhibit development of the degenerative process in jointcartilage or to diminish the degenerative process and thereby improvejoint health in osteoarthritic dogs or in dogs that might otherwisedevelop osteoarthritis. This effect is in addition to anyanti-inflammatory action of omega-3 fatty acids, which may be of lessimportance in canine osteoarthritis because of limited involvement ofinflammation in the osteoarthritis.

Use of an in vitro explant procedure involving articular cartilage asshown in the examples below, demonstrated that, of EPA, DHA and ALA, theonly omega-3 fatty acid to significantly decrease induced release ofglycosaminoglycan (GAG) from the cartilage was EPA. GAGs are astructural component of proteoglycan, therefore, release of GAGindicates degradation of proteoglycan.

With respect to prevention of joint damage from osteoarthritis, aparticular target group of dogs comprises those in-need of suchpreventive care. For example, large breeds such as labrador retriever,rottweiler, German shepherd and the like are more susceptible toosteoarthritis as demonstrated by its greater occurrence in thesebreeds. Additionally, dogs above the age of about 6 years have asignificantly greater occurrence of osteoarthritis. Active dogs,athletic dogs and obese dogs can also be at risk.

The quantity of EPA to be administered can vary substantially. As shownin examples herein, an actual dose response is observed—the greater theamount of EPA administered, the greater the anti-arthritic effect.Generally, a minimum of at least about 0.2% by weight of a nutritiousdiet satisfying ordinary daily requirements of a dog is required. Invarious embodiments, at least about 0.2%, at least about 0.25%, at leastabout 0.3%, at least about 0.4%, at least about 0.5% or at least about0.6% by weight of the diet can be used. Suitably the diet can contain,in various embodiments, up to about 5%, up to about 4%, up to about 3%,up to about 2.5%, up to about 2.25% or up to about 2% by weight EPA. Allpercentages by weight herein, unless otherwise specified, are on a drymatter basis.

A specific amount of EPA can be included in the usual food ration on adaily basis, or the same amount can be provided to the animal in a treator supplement on a daily basis. A combination of these or any otherdosing means can be employed as long as an effective quantity of EPA isprovided daily.

In mixtures of omega-3 and omega-6 fatty acids, the weight ratio ofomega-3 to omega-6 fatty acid can vary significantly. In variousembodiments, the omega-6 to omega-3 weight ratio can be about 1.1:1 toabout 0.2:1 or about 1.08:1 to about 0.42:1; for example about 0.2:1,about 0.25:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about0.8:1, about 1.0:1, or greater. In various embodiments, the omega-6 toEPA weight ratio can be about 12.5:1 to about 1.0:1 or about 12.4:1 toabout 1.12:1, for example about 0.2:1, about 0.25:1, about 0.3:1, about0.4:1, about 0.5:1, about 0.6:1, about 0.8:1, about 1.0:1, about 1.5:1,about 2:1, about 2.5:1, about 3:1, about 4:1, about 5:1, about 6:1,about 7.5:1, about 10:1, about 12.5:1, or greater. In variousembodiments, the arachidonic acid (AA, an omega-6 fatty acid) to EPAweight ratio can be about 2.8:1 to about 0.01:1 or about 0.28:1 to about0.08:1, for example about 0.01:1, about 0.02:1, about 0.04:1, about0.06:1, about 0.08:1, about 1.0:1, about 1.5:1, about 2.8:1, or greater.

The EPA can be administered in amounts calculated as mg/kg body weight.Thus, for example, a 20 kg dog consumes a daily diet of about 275 g offood per day. Amounts of EPA in the diet of about 0.2%, about 0.3%,about 0.4%, about 0.5% or about 0.6% by weight would result inadministering to such a dog about 27.5, about 41.25, about 55, about68.75 or about 82.5 mg/kg body weight respectively. More particularly,EPA can be administered to a dog in an amount of about 20 to about 150mg/kg body weight, for example about 20, about 28, about 30, about 40,about 41, about 50, about 55, about 60, about 69, about 70, about 80,about 82, about 90, about 100, about 120 or about 150 mg/kg body weight,or greater.

Foods are generally classified in the pet food industry as “wet” or“dry”. A wet food has a relatively high amount of water and is usuallypresented in a can or other container wherein air is substantially ortotally excluded. Examples of such foods are “chunk and gravy”compositions, compositions having individual solid particles in thepresence of a liquid gravy, and loaf-type compositions, which generallytake the shape of the container. Dry foods are generally baked orextruded materials, the latter then cut into individual shaped portions,usually known as kibbles. EPA is readily incorporated into a wet foodthrough conventional means. Encapsulation can be employed to protect EPAfrom air oxidation in a dry food. Additionally, use of antioxidants andnitrogen sweeps of packaging can also be employed. This is exemplifiedby U.S. Pat. No. 4,895,725 which has special emphasis onmicro-encapsulation of specific fish oils. Oils which have high levelsof omega-3 fatty acids include oils of menhaden, salmon, cod and thelike.

The present invention also provides, in various embodiments, methodsinvolving administration of a composition comprising EPA to a dog forreducing the severity and frequency of clinical signs of osteoarthritisand the pain associated with this disease without substantial adversereactions or side effects. Additionally, in various embodiments, theinvention provides a method of slowing the clinical progression of anosteoarthritic condition of a dog, the method comprising administrationof a composition comprising EPA. In various embodiments, a method asdescribed herein substantially improves the overall osteoarthriticcondition of the dog so that this benefit can be objectively measuredthrough increased weight bearing in osteoarthritic limbs. The presentinvention also provides methods involving administration of EPA incombination with other treatment modalities for osteoarthritis,including administration of various anti-arthritic medicaments and/orfeeding the animal a weight management diet, both of which are known inthe art.

It is believed that the effect of EPA in decreasing cartilage damageassociated with osteoarthritis can occur at least in part throughdown-regulation of one or more genes responsible for cartilagedegradation. In some cases, one or more genes responsible for cartilagedegradation can be turned off. According to an embodiment of theinvention, mRNA message expression in cartilage tissue of the dog for anenzyme causing cartilage degradation, for example an aggrecanase, isreduced. Decreased cartilage damage can be indicated by a decrease ininduced release of glycosaminoglycan (GAG) from cartilage tissue.

Thus the following are further embodiments of the invention:

A method of down-regulating one or more genes related to enzymaticdegradation of articular cartilage in a dog, the method comprisingadministering to the dog a gene down-regulating effective amount of EPA.

A method for turning off one or more genes related to enzymaticdegradation of articular cartilage in a dog, the method comprisingadministering to the dog a gene turning-off effective amount of EPA.

A method for reducing mRNA message expression in cartilage tissue of adog for an enzyme, e.g., an aggrecanase, causing cartilage degradation,the method comprising administering to the dog an mRNA messageexpression reducing effective amount of EPA.

A method for decreasing induced release of GAG from cartilage tissue ofa dog, the method comprising administering to the dog a GAG releasedecreasing effective amount of EPA.

EXAMPLES

The following examples are merely illustrative, and do not limit thisdisclosure in any way.

Example 1

This example illustrates the release of glycosaminoglycan (GAG) aseffected by omega-3 fatty acids in cultured canine cartilage tissue.

Articular cartilage was obtained from left and right stifles (bothfemoral condyles and tibial plateau) of four dogs. Cartilage explantswere cultured for 3 days in a medium with 10% fetal bovine serum, thenwashed 3 times in a serum-free medium. Explants were then cultured for 6days in a serum-free medium containing 0, 100 or 300 μg/ml n-3 fattyacid (EPA, ALA or DHA). After this period, all explants were washed 3times in a fatty acid-free, serum-free medium. Explants were thencultured individually in triplicate for 4 days in 1 ml fatty acid-free,serum-free medium containing no additives (control, C), 10⁻⁶ M retinoicacid (RA) or 50 ng/ml oncostatin M (OSM). Note that not all treatmentswere possible on all dogs because of cartilage availability. The releaseof GAG into the medium was measured (μg/mg wet weight) at thetermination of culture. In the tables below, the mean and standarddeviation (SD) of GAG release are given for the triplicate cultures fromeach of the four dogs. In addition, the media lactate concentrations(μg/mg wet weight) are given for each treatment. TABLE 1 Results for Dog1 GAG release lactate Treatment n mean SD min max mean SD C 3 1.36 0.500.85 1.84 26.07 33.7 C + carrier 3 1.63 0.31 1.31 1.92 21.95 22.6 C +100 EPA 3 1.59 0.29 1.29 1.87 23.85 25.4 C + 300 EPA 3 1.04 0.53 0.571.61 NA** NA** RA 3 10.50 1.84 8.89 12.50 36.00 39.3 RA + carrier 3 7.154.53 2.00 10.50 33.07 45.4 RA + 100 EPA 3 8.68 2.00 6.61 10.60 29.3734.8 RA + 300 EPA 3 1.59 1.70 0.44 3.54 26.40 39.1 OSM 3 13.60 1.5612.60 15.40 25.37 30.8 OSM + carrier 3 14.25 6.44 7.35 20.10 27.40 33.8OSM + 100 EPA 3 6.29 2.30 4.34 8.80 33.57 52.5 OSM + 300 EPA 3 2.17 1.930.93 4.39 20.05 23.8**not analyzed

As shown in Table 1, significant decrease in GAG release occurred with100 μg/ml EPA in OSM treated cultures and with 300 μg/ml in RA and OSMtreated cultures. There was no significant decrease in media lactateconcentrations with any dose of EPA. TABLE 2 Results for Dog 2 GAGrelease lactate Treatment n mean SD min max mean SD C + carrier 3 0.500.42 0.13 0.96 22.80 NA** C + 100 EPA 3 0.34 0.33 0.10 0.72 39.52 24.57C + 300 EPA 3 0.57 0.46 0.25 1.10 39.20 13.86 OSM + carrier 3 11.70 5.117.10 17.20 26.90 4.77 OSM + 100 EPA 3 5.25 3.00 2.19 8.19 21.70 9.84OSM + 300 EPA 3 2.83 0.23 2.66 3.09 16.23 3.60 C + carrier 3 0.97 0.220.84 1.23 17.40 NA** C + 100 DHA 3 0.64 0.31 0.45 1.00 21.00 6.26 C +300 DHA 3 0.84 0.36 0.43 1.10 36.20 NA** OSM + carrier 3 8.7 0.78 8.109.60 25.33 7.11 OSM + 100 DHA 3 8.57 4.22 3.70 11.20 28.13 2.72 OSM +300 DHA 3 6.07 4.03 3.18 10.70 24.80 1.95 C + carrier 3 0.82 0.68 0.191.55 15.57 1.96 C + 100 ALA 3 1.12 0.09 1.05 1.22 28.40 13.72 C + 300ALA 3 0.99 1.10 0.14 2.24 41.67 14.96 OSM + carrier 3 7.81 7.47 0.2615.20 51.70 28.49 OSM + 100 ALA 3 8.50 4.36 4.09 12.80 28.80 4.96 OSM +300 ALA 3 6.42 2.73 3.44 8.80 55.23 30.31**not analyzed

As shown in Table 2, EPA but not ALA or DHA significantly decreased GAGrelease in OSM treated cultures. There was no significant effect onmedia lactate concentration by any dose of any of the fatty acids. TABLE3 Results for Dog 3 GAG release lactate Treatment n mean SD min max meanSD C + carrier 3 2.73 0.87 2.01 3.69 26.33 4.37 C + 100 ALA 3 2.12 0.431.81 2.61 24.40 4.00 C + 100 DHA 3 1.90 0.83 1.28 2.84 29.35 5.73 C +100 EPA 3 1.67 0.41 1.30 2.11 36.10 NA** C + 300 ALA 3 2.45 0.32 2.142.18 20.75 7.00 C + 300 DHA 3 1.55 0.73 0.73 2.13 28.40 0.57 C + 300 EPA3 1.57 0.39 1.30 2.01 10.53 10.85 RA + carrier 3 20.82 0.65 20.10 21.3738.47 4.78 RA + 100 ALA 3 20.44 0.90 19.40 21.02 43.23 2.28 RA + 100 DHA3 21.09 6.88 13.38 26.60 45.67 8.00 RA + 100 EPA 3 16.22 6.65 8.61 20.9341.53 2.52 RA + 300 ALA 3 24.47 2.99 21.10 26.80 44.73 4.82 RA + 300 DHA3 19.46 2.39 17.28 22.00 47.97 9.14 RA + 300 EPA 3 1.54 0.62 1.08 2.24NA** NA** OSM + carrier 3 12.77 5.85 6.36 17.80 37.87 11.55 OSM − 100ALA 3 22.03 4.60 18.40 27.20 32.77 1.82 OSM − 100 DHA 3 11.67 6.01 5.5017.50 32.27 11.47 OSM − 100 EPA 3 17.85 2.05 16.40 19.30 39.05 11.53 OSM− 300 ALA 3 23.47 3.10 20.30 26.50 34.03 1.38 OSM − 300 DHA 3 11.63 5.076.79 16.90 30.00 5.96 OSM − 300 EPA 3 8.10 6.77 3.79 15.90 21.47 1.93**not analyzed

As shown in Table 3, none of the fatty acids significantly altered GAGrelease from RA- or OSM-stimulated cartilage in this particular animal.There was no change in media lactate associated with any dose of anyfatty acid. TABLE 4 Results for Dog 4 GAG release lactate Treatment nmean SD min max mean SD C + carrier 3 1.96 0.53 1.51 2.55 22.93 4.75 C +100 ALA 3 2.10 0.11 1.98 2.17 20.53 3.48 C + 100 DHA 3 2.34 0.33 2.002.66 19.10 2.35 C + 100 EPA 3 2.69 1.00 1.72 3.71 23.00 6.18 C + 300 ALA3 1.53 1.24 0.13 2.50 29.17 22.07 C + 300 DHA 3 2.31 0.36 1.93 2.6524.93 3.40 C + 300 EPA 3 2.10 0.45 1.64 2.55 24.77 13.00 RA + carrier 314.11 3.89 9.64 16.70 34.53 12.37 RA + 100 ALA 3 12.55 6.35 5.94 18.6039.93 11.59 RA + 100 DHA 3 11.28 7.12 4.79 18.90 25.60 11.77 RA + 100EPA 3 14.39 2.90 11.23 16.93 32.97 4.22 RA + 300 ALA 3 14.09 6.14 8.9820.90 59.37 31.17 RA + 300 DHA 3 11.30 6.82 3.50 16.10 25.33 11.68 RA +300 EPA 3 9.09 1.32 8.26 10.61 25.10 4.67 OSM + carrier 3 16.08 3.5412.05 18.70 31.20 5.99 OSM + 100 ALA 3 11.70 2.19 9.43 13.80 26.33 9.25OSM + 100 DHA 3 24.97 3.26 21.20 26.90 36.83 5.07 OSM + 100 EPA 3 15.884.32 11.95 20.50 27.24 6.34 OSM + 300 ALA 3 19.56 3.91 15.50 23.30 26.676.10 OSM + 300 DHA 3 16.40 6.27 9.40 21.50 36.23 20.34 OSM + 300 EPA 313.49 5.75 7.54 19.02 27.80 2.72

As shown in Table 4, EPA at 300 μg/ml, but not any other fatty acid atany dose, significantly decreased GAG release from RA treated cultures.There was a significant decrease in media lactate concentration incontrol, RA- and OSM-treated cultures with the 300 μg/ml OSMpre-treatment.

Example 2

This example illustrates the incorporation of omega-3 fatty acids intocanine chondrocyte membranes.

The majority of these experiments were performed using monolayercultures, however, in a single experiment, the incorporation of fattyacids into explant cultures of canine cartilage was analyzed.

Monolayer Cultures

Over 24 or 48 hours there was no incorporation of the 18:3 omega-3 fattyacid ALA into chondrocyte membranes from two dogs. The percentage of ALAin chondrocytes incubated in medium alone was <1% out of 5 (range0.3-0.9%) and after 24 or 48 hours of incubation with 100 or 300 μg/mlALA this percentage had not significantly changed (range 0.3-2.5%).

Over 48 hours there was significant incorporation of the 20:5 omega-3fatty acid EPA into chondrocyte membranes from one dog. The percentageof EPA increased from <1% (range 0.2-0.6%) to approximately 7% (range5.6-8%) when cultures were treated with 100 or 300 μg/ml EPA for 48hours. The incorporation was not different when cultures were performedin the presence or absence of 5% fetal calf serum (FCS).

Over 48 hours there was significant incorporation of the 20:5 omega-3fatty acid EPA but not the 18:3 omega-3 fatty acid ALA into chondrocytemembranes from one dog (doses of 300 μg/ml for each fatty acid). Thepercentage of EPA increased from <1% to approximately 15%.

Over 3 or 6 days there was significant incorporation of the 20:5 omega-3fatty acid EPA into chondrocyte membranes from one dog (dose of 300μg/ml EPA). The percentage of EPA increased from <1% to 16-18% with nodifference between 3 and 6 days incubation.

Explant Culture

Over 6 days there was apparent incorporation of the 20:5 omega-3 fattyacid EPA, but not the 18:3 omega-3 fatty acid DHA or the omega-6 fattyacid AA into cartilage explants from one dog (dose of 300 μg/ml for eachfatty acid). The percentage of EPA increased from 0% (none detectable)to approximately 2%.

Summary

These data indicated that EPA, but no other omega-3 fatty acid, wasincorporated into canine chondrocyte membranes in either monolayer orexplant cultures.

Example 3

This example illustrates the effect of omega-3 fatty acids on caninechondrocyte metabolism.

To assess the potential effect of omega-3 fatty acids on protein andproteoglycan metabolism in canine cartilage, cultures were set up asdescribed in Example 1 except for the final 4 days of culture, when nocatabolic stimuli were added (i.e., all “control” cultures). During thefinal 24 hours of culture (i) ³⁵SO₄ (to measure proteoglycan synthesis)or (ii) ³⁵S-methionine and ³⁵S-cysteine (to measure protein synthesis)were added to the medium to radiolabel newly synthesized proteoglycansand proteins, respectively. The incorporation of radiolabel into thecartilage matrix was measured at the termination of culture. No attemptwas made to quantitate loss of radiolabeled material from the cartilageover the 24-hour labeling period. The mean and standard deviation (SD)of the incorporation of 35SO₄ (“PG”) or ³⁵S-methionine and ³⁵S-cysteine(“PROT”) as DPM/mg wet weight are shown in Table 5 below. TABLE 5 PGPROT Treatment n mean SD mean SD Carrier 3 292.7 53.1 574.3 198.3 100ALA 3 246.3 100.8 503.7 184.2 100 DHA 3 156.0 82.5 503.7 81.34 100 EPA 3537.3 161.8 442.0 72.7 300 ALA 3 443.0 205.4 393.7 35.0 300 DHA 3 123.338.2 564.3 220.0 300 EPA 3 275.7 161.7 504.0 44.5

As shown in Table 5, there was no significant effect of any omega-3fatty acid on protein synthesis and incorporation into the matrix. EPAat 100 μg/ml significantly increased proteoglycan synthesis andincorporation. No other dose or fatty acid significantly alteredproteoglycan synthesis and incorporation into the cartilage matrix.

Reverse transcription-PCR was used to measure the mRNA messageexpression levels of matrix proteinases (aggrecanases-1 and -2),cyclooxygenases-1 and -2, lipoxygenases-5 and -12, and potentialautocrine cytokines and their receptors (e.g., IL-1, IL-6 and TNF).

The results of this study found that aggrecanase-1 and aggrecanase-2mRNA messages were expressed in “normal” canine cartilage tissue. Inaddition, some dogs expressed mRNA message of cyclooxygenase-2 (COX-2)message although there were no signs of joint pathology in theseanimals. This enabled monitoring the effects of omega-3 and omega-6fatty acid supplementation on mRNA expression of aggrecanases and COX-2in unstimulated canine articular cartilage explants. EPA was the onlyfatty acid able to reduce the mRNA message for the degradative enzymes,aggrecanase-1 and aggrecanase-2, in canine articular cartilage. Thisdemonstrated the ability of EPA to “turn off” the genes responsible forcartilage degradation.

Example 4

This example illustrates the effects of omega-3 fatty acids in canineosteoarthritis clinical studies.

Three clinical studies were conducted in pet dogs clinically diagnosedwith osteoarthritis. Veterinary general practitioners and orthopedicspecialists enrolled client owned dogs that met specific eligibilitycriteria. All patients were required to (i) have radiographic evidenceof osteoarthritis with measurable clinical manifestations of disease,based on historical accounts by pet owners and physical examinations byveterinarians; (ii) be otherwise healthy and free of concurrent diseasesbased on physical exam, complete blood count (CBC), blood chemistry andurinalysis; and (iii) maintain regimen of therapy if receivingmedications or supplements prescribed for osteoarthritis during the 30days prior to enrolling in the study.

The following measurements were made.

Serum fatty acid profile. This was determined by a gas chromatographymethod involving extraction of fatty acids by chloroform and methanolmixture (2:1), methylation using boron trifluoride-methanol (BF₃:MeOH)reagent followed by flame ionization detection (FID). Fatty acid methylesters were identified by comparison of retention times with those ofknown standards and quantitated using an internal standard.

Veterinary clinical evaluation. Veterinarians conducted both a physicalexam and a clinical evaluation of the patient's osteoarthritic conditionduring the screening phase and at the conclusion of each of the feedingintervals over the course of the clinical trial. Veterinarians assessedthe severity of five osteoarthritic parameters: lameness, reluctance tobear weight, reduction in range of motion, reluctance to hold upcontralateral limb, and pain on palpation of the joint. Changes inseverity scores for these individual parameters were measured over theduration of the feeding period. A comprehensive veterinary clinicalassessment of the impact of dietary intervention on the osteoarthriticcondition of patients was derived by combining the changes in severityscores for all five individual parameters.

Pet owner subjective evaluation. Pet owners were required to complete anenrollment questionnaire prior to participating in the study andadditional questionnaires at the conclusion of each of the feedingintervals over the course of the clinical trial.

-   -   Enrollment questionnaire. Pet owners rated the observed        frequency and severity of the most common signs of canine        osteoarthritis including difficulty rising from rest, limping,        stiffness, soreness when touched, lagging behind during walks,        yelping or whimpering in pain, aggressive behaviors, difficulty        in running, difficulty in walking, difficulty in climbing steps,        difficulty-in jumping, difficulty in playing, impaired mobility,        and overall activity level. In addition, owners rated the        overall osteoarthritic condition of their pet.    -   Feeding questionnaire. Pet owners rated both the frequency and        change in severity of the signs of canine osteoarthritis which        were benchmarked during enrollment. In addition, the pet owners        rated the severity of their animal's pain associated with        osteoarthritis.

Force plate gait analysis. Dogs were evaluated at each institution usinga computerized biomechanics force plate at 0, 6 and 12 weeks. The platewas mounted centrally in and flush with the surface of a 10 m walkway. Ahandler trotted dogs across the force plate and an observer evaluatedeach pass across the plate to confirm foot-strikes and gait. A trial wasconsidered valid if there were distinct ipsilateral fore foot and hindfoot strikes while the dog was trotted across the force plate at avelocity of 1.7-2.0 m/s, with an acceleration variation of −0.5-0.5m/s2. During each trial, the dog's forward velocity was measured, usinga millisecond timer and two photoelectric switches. Each trial wasvideotaped for review and confirmation of valid foot-strikes. Care wastaken to ensure that the dog triggered the timer and that a consistentspeed (as perceived by the handler and observer) was maintained acrossthe plate during each trial.

Five valid trials for each test period were obtained for each affectedlimb and each ipsilateral limb of each dog. Orthogonal ground reactionforces of peak vertical force, vertical impulse, braking and propulsivepeak forces, and braking and propulsion impulses were measured andrecorded by a specialized software program. (Acquire, Sharon Software,DeWitt, Mich.), All forces were normalized with respect to body weightin kilograms. Data from the valid trial for each limb were averaged toobtain a mean value for each force or impulse at each time period.

Ground reaction force data were compared between treatment and placebogroups as a percentage difference between lame and ipsilateral limbs ateach time period. Percentage change of ground force data on the lamelimb were compared at the beginning and end of the feeding period.

Study #1

A canine study was conducted to evaluate the dietary effect of feedingincreased levels of n-3 fatty acids to dogs diagnosed withosteoarthritis. Eighteen veterinary general practitioners were recruitedto enroll patients in the study. A total of 131 dogs were randomlyassigned to two dietary treatments and fed for 180 days. The test andcontrol foods had similar macronutrient profile, but were significantlydifferent in fatty acid composition (Table 6). The test diet containedhigh levels of ALA, EPA and DHA, and was formulated with a low n-6/n-3ratio. The control diet was a leading selling commercially available dogfood, with typical levels of n-3 fatty acids and an n-6/n-3 ratiocharacteristic for the industry. TABLE 6 Dietary nutrient Control food(%) Test food (%) Protein 23.2 19.9 Fat (total) 13.9 13.6 CHO₂ (NFE*)54.7 53.3 ALA (n−3) 0.12 2.8 AA (n−6) 0.03 0.06 EPA (n−3) <0.01 0.38 DHA(n−3) <0.01 0.31 Sum n−6 1.99 2.53 Sum n−3 0.09 3.48 n6/n3 ratio 22.80.7*NFE = Soluble carbohydrate content as nitrogen free extract

Serum fatty acids and pet owner evaluations were recorded at 0, 45, 90and 180 days. Serum fatty acid profiles were significantly modulated bythe test food. The test group had significantly higher concentrations ofn-3 fatty acids (P<0.01), specifically EPA, DHA and ALA, significantlylower concentrations of AA (P<0.01), and significantly lower n-6/n-3ratios (P<0.01) as compared to the control group at the conclusion ofeach feeding interval (Table 7). The test group showed significantimprovements for rising from rest, running and playing at day 45 andwalking at days 90 and 180 as compared to the control group based on petowner observations (P<0.05), even in the presence of a strong placeboeffect (Table 8). TABLE 7 Mean serum fatty acid levels (mg/dl) Group Day0 Day 45 Day 90 Day 180 ALA (n-3) Control 1.10 0.89 0.52 0.53 Test 1.055.61 6.51 7.13 AA (n-6) Control 71.35 66.34 68.03 68.21 Test 64.32 45.9046.13 42.65 EPA (n-3) Control 1.14 0.90 0.67 0.93 Test 1.28 16.28 18.6419.94 DHA (n-3) Control 2.67 2.03 1.70 1.98 Test 2.93 11.31 12.24 12.17Sum n-6 Control 141.08 138.72 137.85 140.28 Test 130.85 118.87 128.71123.99 Sum n-3 Control 4.95 3.84 2.93 3.51 Test 5.36 33.20 37.39 39.24n-6/n-3 ratio Control 33.33 37.95 51.59 51.39 Test 33.90 7.47 8.63 6.92

TABLE 8 Pet owner observed change in severity of osteoarthritis* Day0-45 Day 45-90 Day 90-180 Osteoarthritic P P P sign Group mean valuemean value mean value Rising from Control 1.77 .041 1.77 nsd** 1.93nsd** rest Test 1.56 1.84 1.91 Running Control 1.81 .037 1.83 nsd** 1.94nsd** Test 1.56 1.71 1.91 Walking Control 1.71 nsd** 2.00 .018 2.19 .002Test 1.69 1.71 1.75 Playing Control 1.83 .008 1.90 nsd** 2.06 nsd** Test1.50 1.78 1.97*Osteoarthritis severity rating scale: 1 = better, 2 = no change, 3 =worsened.**nsd = no significant difference.Study #2

A canine study was conducted to evaluate the dietary effect of feedingincreased levels of n-3 fatty acids to dogs diagnosed withosteoarthritis. Two veterinary orthopedic specialists enrolled patientsin the study. A total of 38 dogs were randomly assigned to two dietarytreatments and fed for 90 days. The test and control diets weremanufactured from the same lots of foods as described above (Table 6).

Serum fatty acids, force plate gait analysis, and veterinary clinicalassessments were recorded at 0, 45 and 90 days. Serum fatty acidprofiles were significantly modulated by the test food. The test grouphad significantly higher serum concentrations of n-3 fatty acids(P<0.01), specifically EPA, DHA and ALA, significantly lowerconcentrations of AA at day 90 (P<0.01), and significantly lower n-6/n-3ratios (P<0.01) as compared to the control group at the conclusion ofeach feeding interval (Table 9). TABLE 9 Mean serum fatty acid levels(mg/dl) Day 0 Day 45 Day 90 Group mean P value mean P value mean P valueALA (n-3) Control 0.89 0.7764 0.34 <0.0001 0.27 <0.0001 Test 0.98 4.455.04 AA (n-6) Control 55.55 0.6880 50.78 0.0736 55.95 0.0001 Test 57.1341.94 38.01 EPA (n-3) Control 1.19 0.7000 0.34 <0.0001 0.20 <0.0001 Test1.54 11.52 11.89 DHA (n-3) Control 4.30 0.4323 1.82 <0.0001 1.32 <0.0001Test 3.37 11.15 11.21 Sum n-6 Control 122.85 0.2508 112.46 0.0148 114.600.0036 Test 113.61 91.72 89.85 Sum n-3 Control 6.36 0.8335 2.57 <0.00011.79 <0.0001 Test 5.90 27.14 28.13 n-6/n-3 ratio Control 32.54 0.252166.66 <0.0001 75.90 <0.0001 Test 45.90 8.48 3.59

A biomechanical assessment of the dogs' most severe osteoarthritic limbwas objectively evaluated using force plate gait analysis (Table 10).Vertical peak force is the key parameter measured to determine weightbearing of the affected limb. There was no significant change in meanvertical peak force over the duration of the 90 day feeding for thecontrol group (P=0.91), while there was a significant increase in meanvertical peak force over time for the test group (P=0.01). The percentmean change in vertical peak force was also significantly differentbetween groups (P<0.05), indicating that the test group increased weightbearing in the affected limb, while the control group displayed nochange in weight bearing over the course of the study. Weight bearingability can also be represented by displaying the frequency distributionof percent change in vertical peak for each dietary group. Only 31% ofanimals in the control group showed improvement in weight bearing afterthe 90 day feeding, while 82% of the dogs in the test group increasedweight bearing over the course of the study. TABLE 10 Vertical peakforce Day 0 Day 90 Change (Day 0-90) P P mean mean = 0 % mean Group meanvalue mean value change Pr > l t l change Pr > l t l Control 72.800.5981 72.63 0.9323 −0.17 0.9144 −0.58 0.0443 Test 69.51 73.21 3.710.0103 5.35

The subjective clinical evaluations performed by the veterinaryorthopedic surgeons provided additional support for the efficaciousnessof the test diet. Based upon the comprehensive veterinary clinicalassessment, a significantly greater percent of dogs were evaluated asimproved that consumed the test food as compared to dogs that consumedthe control food (P<0.05). The veterinary specialists also observed agreater percent of dogs in the test group displaying a reduction in painon palpation of the joint as compared to the control group (P=0.05).

Study #3

A canine study was conducted to determine the dose effect of feedingincreased levels of n-3 fatty acids to dogs diagnosed withosteoarthritis. Twenty-eight veterinary general practitioners enrolledpatients in the study. A total of 177 dogs were randomly assigned tothree dietary treatments and fed for 90 days. Approximately two-thirdsof the dogs participating in the study were receiving medications and/orsupplements prescribed for treating osteoarthritis, in addition toconsuming the therapeutic diets being evaluated. The three test foodshad similar macronutrient profiles, but varied in composition of EPA andDHA, with variable A containing the lowest levels and variable Ccontaining the highest levels (Table 11). TABLE 11 Test variable %Dietary nutrient A B C Protein 19.97 19.51 19.37 Fat (total) 13.78 15.3419.55 CHO₂ (NFE*) 53.92 52.34 47.66 ALA (n−3) 2.65 1.18 1.10 AA (n−6)0.11 0.18 0.24 EPA (n−3) 0.50 1.18 1.69 DHA (n−3) 0.34 0.80 1.15 Sum n−62.70 2.45 2.14 Sum n−3 3.54 3.53 4.52 n−6/n−3 ratio 0.76 0.70 0.47*NFE = Soluble carbohydrate content as nitrogen free extract

Serum fatty acids, pet owner evaluations, and veterinary clinicalassessments were recorded at 0, 21, 45 and 90 days. Serum fatty acidprofiles were significantly modulated by all dietary variables. The dogsfed test variables B & C had significantly higher serum concentrationsof n-3 fatty acids (P<0.01), specifically EPA, DHA and ALA,significantly lower concentrations of n-6 fatty acids, specifically AA(P<0.01), and significantly lower n-6/n-3 ratios (P<0.01) as compared tothe dogs fed test variable A at the conclusion of each feeding interval(Table 12). TABLE 12 Mean serum fatty acid levels (mg/dl) Test variableDay 0 Day 21 Day 45 Day 90 ALA (n-3) A 1.34 5.65 5.29 5.63 B 1.29 3.363.99 3.82 C 1.25 2.92 3.32 3.29 AA (n-6) A 76.37 51.10 47.54 47.77 B73.15 41.55 38.94 37.00 C 70.05 37.35 36.86 34.73 EPA (n-3) A 1.32 18.7418.51 19.26 B 1.54 26.14 29.87 30.03 C 1.85 34.42 35.71 39.04 DHA (n-3)A 3.50 13.75 13.84 13.88 B 4.72 18.47 19.98 20.16 C 3.91 21.01 21.4722.49 Sum n-6 A 150.38 114.38 110.12 112.70 B 143.93 93.83 95.87 92.10 C139.97 79.71 82.65 80.74 Sum n-3 A 6.16 38.14 37.65 38.77 B 7.55 47.9653.84 54.01 C 7.01 58.35 60.50 68.83 n-6/n-3 ratio A 29.99 5.65 3.483.75 B 28.09 3.36 1.92 1.79 C 32.30 2.92 2.02 1.73

Pet owners reported improvements in 13 of 14 individual osteoarthriticsigns for dogs consuming any of the dietary variables for 21 days (Table13). Additionally, pet owners reported a decrease in severity for 13 of14 individual osteoarthritic signs for dogs consuming any of the dietaryvariables for 90 days (Table 14). Pet owners also reported a significantreduction in the frequency of observable osteoarthritic signs after thedogs consumed any of the dietary variables for 90 days (Table 15). TABLE13 Pet owner observed improvements in osteoarthritic signs (Day 0-21)Osteoarthritic Mean = 0 Osteoarthritic Mean = 0 Sign Diet Mean Pr > l tl Sign Diet Mean Pr > l t l Rising from A −0.439 0.0002 Running A −0.5240.0004 rest B −0.738 <0.0001 B −0.682 <0.0001 C −0.763 <0.0001 C −0.674<0.0001 Limping A −0.720 <0.0001 Walking A −0.553 0.0007 B −0.731<0.0001 B −0.750 <0.0001 C −0.837 <0.0001 C −0.667 <0.0001 Stiffness A−0.537 <0.0001 Stair A −0.449 0.0012 B −0.783 <0.0001 climbing B −0.667<0.0001 C −0.627 <0.0001 C −0.723 <0.0001 Soreness A −0.750 0.0005Jumping A −0.362 0.0049 B −0.800 0.0002 B −0.600 <0.0001 C −0.379 0.0451C −0.542 <0.0001 Lagging A −0.564 0.0004 Playing A −0.622 <0.0001 behindon B −0.909 <0.0001 B −0.763 <0.0001 walks C −0.531 0.0022 C −0.4870.0014 Pain A −0.476 0.0245 Impaired A −0.528 0.0005 B −0.478 0.0184mobility B −0.700 <0.0001 C −0.889 0.0002 C −0.564 <0.0001 Aggression A0.000 1.0000 Activity A −0.745 <0.0001 B −0.313 0.1050 level B −0.857<0.0001 C −0.429 0.1401 C −0.865 <0.0001

The above “p” values refer to the mean change from day 0 to day 21.TABLE 14 Difference in pet owners' severity rating (Day 0-90)Osteoarthritic Osteoarthritic Sign Diet Mean Pr > t Sign Diet Mean Pr >t Rising from rest A −0.463 <0.0001 Running A −0.579 <0.0001 B −0.633<0.0001 B −0.558 <0.0001 C −0.518 <0.0001 C −0.605 <0.0001 Limping A−0.489 0.0003 Walking A −0.294 0.0358 B −0.588 <0.0001 B −0.643 <0.0001C −0.681 <0.0001 C −0.595 <0.0001 Stiffness A −0.255 0.0420 Stair A−0.419 0.0024 B −0.483 <0.0001 climbing B −0.489 0.0002 C −0.589 <0.0001C −0.689 <0.0001 Soreness A −0.810 <0.0001 Jumping A −0.571 0.0003 B−0.920 <0.0001 B −0.479 0.0011 C −0.926 <0.0001 C −0.773 <0.0001 LaggingA −0.657 <0.0001 Playing A −0.606 0.0002 behind on B −0.531 0.0014 B−0.571 0.0003 walks C −0.448 0.0094 C −0.694 <0.0001 Pain A −0.6840.0002 Lameness A −0.484 0.0045 B −0.571 0.0009 B −0.778 <0.0001 C−0.667 0.0010 C −0.667 <0.0001 Aggression A −0.750 0.0234 Activity A−0.409 0.0009 B −1.000 0.0025 level B −0.704 <0.0001 C −1.000 0.0751 C−0.551 <0.0001

The above “p” values refer to the mean change form day 0 to 90. TABLE 15Difference in pet owners' frequency rating (Day 0-90) OsteoarthriticOsteoarthritic Sign Diet Mean Pr > t Sign Diet Mean Pr > t Rising fromrest A −0.370 <0.0001 Limping A −0.239 <0.0165 B −0.467 <0.0001 B −0.365<0.0001 C −0.509 <0.0001 C −0.396 <0.0001 Stiffness A −0.098 0.2929Lagging A −0.571 <0.0001 B −0.373 <0.0001 behind on B −0.643 <0.0001 C−0.421 <0.0001 walks C −0.500 0.0004 Soreness A −0.381 0.0146 AggressionA −0.417 0.0536 B −0.680 <0.0001 B −0.467 0.0175 C −0.821 <0.0001 C−0.167 0.5741 Running A −0.447 0.0004 Walking A −0.206 0.0911 B −0.3950.0009 B −0.558 <0.0001 C −0.477 <0.0001 C −0.447 0.0002 Jumping A−0.357 0.0027 Stair A −0.302 0.0069 B −0.354 0.0015 climbing B −0.3480.0014 C −0.467 <0.0001 C −0.457 <0.0001 Playing A −0.455 0.0013Impaired A −0.250 0.0643 B −0.297 0.0238 mobility B −0.436 0.0005 C−0.667 0.0010 C −0.667 <0.0001

Dogs consuming higher concentrations of n-3 fatty acids were reported tohave more significant improvement in osteoarthritic condition and moresignificant reduction in the progression of osteoarthritis than thosedogs receiving the lowest dosage, based on veterinarians clinicalassessments (Table 16). There was no significant difference inimprovement in osteoarthritic condition or reduction in the progressionof osteoarthritis between the group receiving medications and/orsupplements and the non-medicated group (Table 17). This indicates thatthe therapeutic diets work synergistically with other therapies or atleast not withstanding other therapies by providing additional benefitto dogs suffering from osteoarthritis.

An extremely low incidence of adverse reactions or side effects wasreported among dogs participating in this study. Only five dogs out ofthe 215 animals assigned to food were reported to have diarrhea andvomiting, which could possibly be attributed to consuming one of thedietary variables. Similar incidence of adverse reactions or sideeffects were reported for those dogs consuming the therapeutic diets inthe previous two studies discussed (1/88 and 1/26 for examples 1 and 2respectively). TABLE 16 Progression of Overall change in osteoarthriticcondition osteoarthritic condition Diet n mean P Diet n mean P A 552.327 0.2891 A vs A 54 3.148 0.1675 A vs B B B 62 2.177 0.1619 B vs B 622.871 0.0787 B vs C C C 59 1.983 0.0168 A vs C 59 2.525 0.0024 A vs C C

TABLE 17 Overall change in Progression of osteoarthritic conditionosteoarthritic condition Med- Medi- i- Diet cated n mean P Diet cated nmean P A no 22 2.273 0.6665 A no 21 3.143 0.9770 yes 33 2.364 yes 333.152 B no 23 2.130 0.7109 B no 23 2.696 0.3247 yes 39 2.205 yes 392.974 C no 28 2.071 0.4003 C no 28 2.750 0.1285 yes 31 1.903 yes 312.323

All patents and publications cited herein are incorporated by referenceinto this application in their entirety.

The words “comprise”, “comprises”, and “comprising” are to beinterpreted inclusively rather than exclusively.

1. A method for increasing mobility of a dog having arthritis, themethod comprising administering to the dog eicosapentaenoic acid in anamount effective to decrease cartilage damage in the dog.
 2. The methodof claim 1, wherein one or more genes responsible for cartilagedegradation are down-regulated.
 3. The method of claim 1, wherein one ormore genes responsible for cartilage degradation are turned off.
 4. Themethod of claim 1, wherein mRNA message expression in cartilage tissueof the dog for an enzyme causing cartilage degradation is reduced. 5.The method of claim 4, wherein the enzyme is an aggrecanase.
 6. Themethod of claim 1, wherein induced release of glycosaminoglycan fromcartilage tissue of the dog is decreased.
 7. A method for increasingweight bearing in a limb of a dog having arthritis, the methodcomprising administering to the dog eicosapentaenoic acid in an amounteffective to decrease cartilage damage in the dog.
 8. The method ofclaim 7, wherein one or more genes responsible for cartilage degradationare down-regulated.
 9. The method of claim 7, wherein one or more genesresponsible for cartilage degradation are turned off.
 10. The method ofclaim 7, wherein mRNA message expression in cartilage tissue of the dogfor an enzyme causing cartilage degradation is reduced.
 11. The methodof claim 10, wherein the enzyme is an aggrecanase.
 12. The method ofclaim 7, wherein induced release of glycosaminoglycan from cartilagetissue of the dog is decreased.
 13. A method for reducing painassociated with arthritis in a dog, the method comprising administeringto the dog eicosapentaenoic acid in an amount effective to decreasecartilage damage in the dog.
 14. The method of claim 13, wherein one ormore genes responsible for cartilage degradation are down-regulated. 15.The method of claim 13, wherein one or more genes responsible forcartilage degradation are turned off.
 16. The method of claim 13,wherein mRNA message expression in cartilage tissue of the dog for anenzyme causing cartilage degradation is reduced.
 17. The method of claim16, wherein the enzyme is an aggrecanase.
 18. The method of claim 13,wherein induced release of glycosaminoglycan from cartilage tissue ofthe dog is decreased.
 19. A method for modulating enzymatic degradationof articular cartilage in a dog, the method comprising administering tothe dog an enzymatic degradation modulating effective amount ofeicosapentaenoic acid.
 20. The method of claim 19, wherein one or moregenes responsible for cartilage degradation are down-regulated.
 21. Themethod of claim 19, wherein one or more genes responsible for cartilagedegradation are turned off.
 22. The method of claim 19, wherein mRNAmessage expression in cartilage tissue of the dog for an enzyme causingcartilage degradation is reduced.
 23. The method of claim 22, whereinthe enzyme is an aggrecanase.
 24. The method of claim 22, wherein thereduction of mRNA message expression is accompanied by incorporation ofeicosapentaenoic acid into membranes of chondrocytes.
 25. The method ofclaim 19, wherein induced release of glycosaminoglycan from cartilagetissue of the dog is decreased.
 26. The method of claim 19, wherein theeicosapentaenoic acid is administered orally in a food composition. 27.The method of claim 26, wherein the food composition comprises at leastabout 0.2% by weight eicosapentaenoic acid on a dry matter basis. 28.The method of claim 26, wherein the food composition is a component of anutritional diet, a snack, a supplement or a treat.
 29. The method ofclaim 19, wherein the eicosapentaenoic acid is administered orally in anutraceutical or pharmaceutical dosage form.